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Infection and Immunity, September 2001, p. 5278-5285, Vol. 69, No. 9
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.9.5278-5285.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
vsp Gene Expression by
Giardia lamblia Clone GS/M-83-H7 during Antigenic
Variation In Vivo and In Vitro
M.
Bienz,
M.
Siles-Lucas,
P.
Wittwer, and
N.
Müller*
Institute of Parasitology, University of
Berne, Berne, Switzerland
Received 27 January 2001/Returned for modification 5 May
2001/Accepted 29 May 2001
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ABSTRACT |
Giardia lamblia infections are associated with
antigenic variation of the parasite, which is generated by a continuous
change of the variant-specific surface proteins (VSPs). Many
investigations on the process of antigenic variation were based on the
use of G. lamblia clone GS/M-83-H7, which expresses VSP
H7 as its major surface antigen. In the present study, mice were
infected with the aforementioned clonal line to investigate
vsp gene expression during the complex process of
antigenic variation of the parasite. Trophozoites collected from the
intestines of individual animals at different time points postinfection
(p.i.) were analyzed directly for their vsp gene
expression patterns, i.e., without cultivating the recovered parasites
in vitro. Because few trophozoites were recovered at late time points
p.i., a combined 5' rapid amplification of cDNA ends-reverse
transcription-PCR approach was utilized. This allowed detection
and subsequent sequence analysis of vsp gene transcripts
upon generation of amplified cDNA analogues. The same PCR approach was
applied for analysis of vsp gene expression in variants
obtained after negative selection of axenic GS/M-83-H7 trophozoites by
treatment with a cytotoxic, VSP H7-specific monoclonal antibody. In an
overall view of the entire panel of in vivo- and in vitro-derived
parasite populations, expression of 29 different vsp
gene sequences could be demonstrated. In vivo antigenic variation of
G. lamblia clone GS/M-83-H7 was shown to be a continuous
process involving the consecutive appearance of relatively distinct
sets of vsp transcripts. During the 42-day infection
period investigated, this process activated at least 22 different
vsp genes. Comparative molecular analyses of the amino
acid level demonstrated that all cDNA segments identified encode
structural elements typical of the terminal segment of
Giardia VSP. The similarity of most of the GS/M-83-H7
VSP sequences identified in the present study supports previous
suggestions that vsp gene diversification in G.
lamblia is the result of ancestral gene duplication, mutation,
and/or recombination events.
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INTRODUCTION |
The protozoan parasite Giardia
lamblia is an important causative agent of acute or chronic
diarrhea in humans and various animals. G. lamblia is
characterized by a considerable potential to alter its surface
antigens, a phenomenon that is mediated by a unique protein family, the
variant-specific surface proteins (VSPs) (19). Individual
trophozoites basically produce a single VSP, which constitutes the
major surface antigen and which covers the entire cell including the
flagella (27). The proteins are cysteine rich, exhibiting
a common four-amino-acid CXXC repeat motif and remarkable similarity
across the hydrophobic, membrane-spanning region at the extreme C
terminus (15, 26).
The antigenic surfaces of different isolates of G. lamblia
are basically variable (23, 25, 34), and surface antigenic variation may occur within an isolate (1-3, 20, 21).
Surface antigen alterations have been observed within proliferating
trophozoite populations inside the host (3, 7, 8, 32), as
well as among trophozoites upon release from nonproliferative cysts (13, 33). This change of the surface protein coat is
supposed to allow the parasite to persist within the hostile
immunological and physiological environment of the gut (reviewed in
references 16 and 18). Since a maturating humoral immune response in both experimental and natural Giardia-infected hosts
coincides with the elimination of the original variant phenotype, a
functional role of antibodies in the selection of variants was proposed
(11, 21). Support for this conclusion was provided by the
finding of a parasiticidal effect of anti-Giardia antibodies
on in vitro-cultivated trophozoites. Thus, incubation of G. lamblia trophozoites with sera from infected individuals
(11, 21) and with monoclonal antibodies (MAbs) to
different VSPs (21) resulted in a complement-dependent lysis of the parasites. Furthermore, a MAb (G10/4) to VSP H7
(2) produced by G. lamblia clone GS/M-83-H7 and
a MAb (6E7) to VSPA6 produced by G. lamblia isolate WB
(20) induced complement-independent lysis of the
trophozoites with respective variant antigen types (9, 20,
31). High concentrations of MAbs G10/4 (31) and 6E7
(20) caused immediate immobilization as well as detachment and aggregation of the trophozoites. The direct cytotoxic effect of MAb
G10/4 was associated with shedding VSP-containing membrane vesicles
from the parasite surface and a concomitant partial disruption of the
cellular membrane (9).
So far, the process of antigenic variation of G. lamblia in
vivo and the host's immune response against the parasite have best
been studied in experimentally infected mice (reviewed in references 5
and 16). Investigations have been mostly based on the use of G. lamblia clone GS/M-83-H7, which expresses major surface antigen
VSP H7 (2). Experimental G. lamblia GS/M-83-H7 infections in a combined mother-offspring mouse model involving simultaneous infection of mother and offspring animals indicated that
ingestion of anti-VSP H7 secretory immunoglobulin A antibodies by
neonatal mice is associated with an immediate increase in the prevalence of new variant antigen types within the intestinal parasite
population of these animals (32). These antibodies display
a direct cytotoxic effect against VSP H7-type trophozoites (32). We have now investigated in detail at the
transcriptional level the complexity of the antigenic diversification
process during an experimental infection with G. lamblia
clone GS/M-83-H7 in the murine host. Antigenic variation in vivo was
found to be associated with activation of a large portion of the
vsp gene repertoire of the parasite. A remarkable
diversification in vsp gene expression was also observed in
vitro upon antibody (MAb G10/4)-mediated growth selection for VSP
H7-negative trophozoites within a culture of G. lamblia
clone GS/M-83-H7 trophozoites.
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MATERIALS AND METHODS |
Mice.
Gravid 10- to 12-week-old outbred CD-1(ICR)BR mice
were obtained from Charles River GmbH, Germany. Animals were
kept according to the Swiss regulations of animal experiments with free
access to germ-free food and sterile water.
Parasite.
The origin, axenization, and cloning of G. lamblia clone GS/M-83-H7 have been described by Aggarwal and
coworkers (2). This clone expresses on its surface a major
72-kDa antigen (VSP H7), which is recognized by MAb G10/4. Trophozoites
from a clonal GS/M-83-H7 line of G. lamblia were cultivated
in modified TYI-S-33 medium with antibiotics as previously described
(12).
Immunofluorescence assays.
The kinetics of expression of the
major surface antigen on trophozoites isolated from the duodena of
experimentally infected mice (see below) were assessed by
immunofluorescence using MAb G10/4 as described previously
(7).
Experimental parasite infection, sample collection, and total RNA
extraction.
Three-day-old offspring and the respective mothers
were infected with G. lamblia trophozoites (clone
GS/M-83-H7) as described previously (32).
The course of the G. lamblia infection within offspring was
determined as described by Gottstein and coworkers (6) by
quantifying the parasite burden through microscopical examination of
adherent trophozoites from intestinal washes.
For total RNA extraction (see below), intestinal parasites were
isolated as follows. Sections of about 1 cm from the upper part
of the duodenum were slit longitudinally and subsequently incubated for
20 min in 1 ml of phosphate-buffered saline (PBS; containing 0.15 M
NaCl), pH 7.2, on ice to detach trophozoites from the intestinal
surface. Then, 0.5 ml of PBS supernatant containing detached
trophozoites was transferred into a well of a microtiter plate
(Cellstar TC plate; 24 wells; Greiner Labortechnik GmbH, Frickenhausen,
Switzerland), and viable parasites were allowed to adhere to the bottom
of the well by incubation for 20 min at 37°C. After the parasites
were washed three times with 1 ml of prewarmed (37°C) PBS, residual,
adherent trophozoites were resuspended in 100 µl of lysis
buffer-
-mercaptoethanol (
-ME) mixture from the StrataPrep
Total RNA Microprep kit (Stratagene, La Jolla, Calif.). The lysates
were processed for extraction of total RNA as instructed including
treatment with RNase-free DNase I. Finally, total RNA preparations
(solubilized in 30 µl of elution buffer) were stored at
80°C
until further used.
Antigen switching by GS/M-83-H7 trophozoites in vitro, sample
collection, and total-RNA extraction.
The procedure used to detect
antigen switching of in vitro-cultivated G. lamblia clone
GS/M-83-H7 trophozoites from a VSP H7-positive to a VSP H7-negative
parasite population was adapted from a method of Nash et al.
(21). Adherent GS/M-83-H7 trophozoites from late-log-phase
cultures were washed twice in prewarmed (37°C) PBS and then detached
from the bottom of the culture tube by a 15-min incubation in ice-cold
PBS. About 105 cells (corresponding to 200 µl
from the resulting parasite suspension) were transferred into a well
from a culture plate and subsequently allowed to readhere for 15 min at
37°C. To select against VSP H7-positive trophozoites within this
parasite population, incubation was continued for 15 min after adding 2 µl of ascites fluid containing cytotoxic, VSP H7-specific MAb
G10/4. Nonadherent, dead trophozoites were removed by washing three
times with 1 ml of prewarmed (37°C) PBS, after which the residual
adherent (MAb G10/4-resistant) variant trophozoites were detached in
500 µl of ice-cold PBS. Released parasites were grown in vitro for 3 days in 15 ml of TYI-S-33 medium and then re-treated with MAb
G10/4 and recultivated as described above. After confirmation by
immunofluorescence that the resulting MAb G10/4-resistant parasite
population was essentially (>99%) VSP H7 negative, about
104 of these negatively selected variant
trophozoites (<1% VSP H7 positive) treated with MAb G10/4 were
resuspended in 100 µl of a lysis buffer-
-ME mixture from the
StrataPrep Total RNA Microprep kit (Stratagene). In parallel, an
analogous cellular lysate was prepared from about
104 GS/M-83-H7 trophozoites that had not been
treated with MAb G10/4. Both cellular lysates were then further
processed for total-RNA extraction as described above. Finally,
total-RNA preparations were solubilized in 30 µl of elution buffer
and stored at
80°C until further used.
Analysis of vsp gene transcripts by reverse
transcription-PCR.
cDNA was synthesized by reverse transcription
from total RNA, prepared from in vitro-cultivated and intestinal
trophozoite populations, by using an oligo(dT) primer, avian
myeloblastosis virus reverse transcriptase, and other components of the
5'-to-3' rapid amplification of cDNA ends (RACE) kit (Roche Diagnostics GmbH, Rotkreuz, Switzerland) (Fig. 1A,
step 1) as instructed. The same kit system was used for 5' RACE (Fig.
1A, steps 2 and 3) by the addition of a 3' poly(dA) tail to the 3' end
of the cDNA and a subsequent amplification (PCR-1; 40 cycles of 94°C for 30 s, 38°C for 30 s, and 72°C for 150 s, ending
with a 15-min extension at 72°C) of the tailed cDNA using the kit's
oligo(dT) as the forward primer (complementary to the artificially
introduced poly(dA) tail; see above) and vsp gene-specific
reverse primer vsp-rev1
(5'-CGGCGGCCGCTCAGAACCACCAGCAGAGGAA-3')
(Fig. 1B). The latter contained a NotI restriction site
(underlined) as an anchor sequence, including the stop codon (in
boldface), and a sequence complementary to nucleotides (nt) 1636 to
1653 (in italics) of the vsp H7 gene. This
vsp-specific PCR occurred as a seminested reaction
concomitant with general "nonspecific" single-primer amplification
of all cDNA by oligo(dT) priming (Fig. 1A, steps 3.1 and 3.2). Finally,
selective amplification of vsp cDNA molecules (PCR-2; 5 cycles of 94°C for 30 s, 38°C for 30 s, and 72°C for
150 s and then 30 cycles of 94°C for 30 s, 60°C for
30 s, and 72°C for 150 s, with a final 15-min extension at 72°C) was achieved using degenerate vsp gene-specific
forward primer vsp-forward
(5'-GCAGATCTCACIAAYGGIGTITGYACIGC-3,
where I = deoxyinosine and Y = C or T) containing a Bgl2
restriction site (underlined) followed by nt 1147 to 1166 (in italics)
of the vsp H7 gene and reverse anchor primer
vsp-rev2
(5'-CGGCGGCCGCTCAGAACCAC-3'), representing
the first 20 nt from the vsp-rev1 primer, used for PCR-1
(Fig. 1A, step 4, and B). Introduction of artificial mutations during
amplification was minimized by using the Expand High Fidelity PCR
system (Roche Diagnostics) for all PCRs. Controls were included for
both PCR steps using total RNA equivalents that had not been reverse
transcribed into cDNA (not shown) to check that no DNA was amplified
from any residual genomic DNA that might have resisted DNase I
digestion. PCR products were analyzed by electrophoresis in 2% agarose
gels. The amplification products from the predominant band (migrating
with a size of around 550 bp) were tested for restriction fragment
length polymorphism (RFLP) by cleavage with AluI.
Additionally, the amplification products were cloned into the pGEMeasy
vector (Promega, Madison, Wis.), and cloned inserts were sequenced by a
commercial sequencing service (Solvias AG, Basel, Switzerland). All
recombinant DNA methods, unless otherwise stated, were those of
Sambrook et al. (29). For DNA cloning, Escherichia
coli XL1-Blue (Stratagene) was used as the bacterial host.

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FIG. 1.
Strategy used for PCR-based amplification and analysis
of vsp cDNA molecules. (A) In step 1, polyadenylated
(lowercase) mRNA present within total trophozoite RNA was reverse
transcribed into cDNA using an oligo(dT) primer (italic uppercase). In
step 2, the first-strand cDNA molecules were polyadenylated (boxed
uppercase) at the 3' end using terminal transferase. In step 3, 3'-polyadenylated cDNA was amplified by PCR using oligo(dT) as the
forward and reverse primers (step 3.1). This increased the amount of
cDNA template available for amplification of vsp cDNA
mediated by oligo(dT) (as the forward primer) and reverse
vsp-specific (open box) primer vsp-rev1
(step 3.2; see also panel B), containing a short anchor
sequence (grey box). In step 4, the heterogeneous cDNA amplified in
step 3 was used as a template for subsequent nested PCR, which allowed
more-specific amplification using degenerate forward vsp
primer vsp-forward (open box) and reverse primer
vsp-rev2, modified from vsp-rev1 (see
also panel B). (B) Nucleotide positions of the vsp H7
target sequences from primers vsp-forward,
vsp-rev1, and vsp-rev2. Primer
vsp-forward contains a 5' GC, followed by a Bgl2
restriction site (underlined) as the anchor sequence and
a degenerate sequence targeting between nt 1147 and 1166 (italics) of
the vsp H7 gene (I = deoxyinosine; Y = C or
T). Primer vsp-rev1 contains a 5' GC, a
NotI restriction site (underlined), and the stop codon
(boldface) as the anchor sequence and a sequence
complementary to nt 1636 to 1653 (italics) of the vsp H7
gene. Reverse primer vsp-rev2 represents the first 20 nt
from vsp-rev1.
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Sequence alignments.
Alignment and comparison of the
corresponding amino acid sequences derived from the studied in vivo-
and in vitro-derived parasite populations, including the original
GS/M-83-H7 isolate, were done using the MultAlin and the ESPript1.9
computer software, available at the ExPASy molecular biology server
using the Blosum62 symbol comparison table (10). The newly
derived sequences were aligned against all homologous regions of those
G. lamblia VSP sequences available in GenBank using the
ClustalX, version 1.8, and the Risler symbol comparison
table (28). A gene tree, based on distances
calculated from amino acid sequence alignments by ClustalX, version
1.8, was generated using TreeView, version 1.6.1.
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RESULTS |
Detection of antigen switching in G. lamblia
trophozoites cultivated in vitro.
To select for new variants
within clonal line GS/M-83-H7 of G. lamblia, originally
consisting of more than 95% VSP H7-type (MAb G10/4-positive)
trophozoites, cultivated parasites were treated twice with VSP
H7-specific, cytotoxic MAb G10/4. The efficacy of this selection was
assessed by immunofluorescence, which demonstrated that the treated
culture contained more than 99% non-VSP H7-type (MAb G10/4-negative) trophozoites.
Course of G. lamblia infection in mice and in vivo
antigen switching of the parasite.
In our study, 3-day-old
suckling mice were infected by intragastric injection with trophozoites
from clonal line GS/M-83-H7. At different time points during (days 7 and 14 postinfection [p.i.]) and after (days 21 and 42 p.i.) the
lactation phase, offspring individuals were sacrificed by
CO2 euthanasia. The follow-up study of the
parasite burden confirmed previously published results (17) in that it revealed high infection intensities at
days 7 and 14 p.i. but a significant regression of infection by
day 21 p.i. (data not shown). At day 42 p.i., intestinal
trophozoites had nearly disappeared and only one of the three animals
tested contained a parasite burden sufficient for further
investigations (see below). Immunofluorescence analysis demonstrated
that trophozoites from both the inoculum (representing day 0 p.i.)
and the parasites isolated at day 7 p.i. were mostly (>95%) VSP
H7 positive. In contrast, the intestinal parasite populations recovered
after day 7 p.i., i.e., on days 14, 21, and 42 p.i., did not
possess detectable numbers of VSP H7-type trophozoites in any of the
respective animals tested.
Characterization of cloned vsp cDNA derived from
variants emerging during antigen switching of the parasites in vitro
and in vivo.
For molecular analysis of vsp gene
expression in different G. lamblia clone GS/M-83-H7
populations during antigen switching in vitro or in vivo, trophozoites
were sampled either from (i) an original VSP H7-type GS/M-83-H7 culture
(which represents both the nontreated culture prior to detectable
antigen switching and the parasite inoculum corresponding to day 0 of
the experimental infection; see below), (ii) a culture treated with
cytotoxic MAb G10/4, or (iii) the intestines of individual animals at
days 7, 14, 21, and 42 p.i. From these different sample groups,
total RNA was prepared and then used as a template for cDNA synthesis (Fig. 1A, step 1). To avoid ex vivo antigenic drift of the intestinal parasite populations, sampled trophozoites were further processed without previous proliferation of the parasites through in vitro cultivation. Since sampling of parasites from late-stage infected animals (at days 21 and 42 p.i.) provided only trace amounts of parasite material, cDNA containing vsp gene segments had to
be amplified in several PCR steps prior to cloning and subsequent analysis (Fig. 1A, steps 2 to 4). The final PCR, specifically amplifying vsp cDNA, was performed using a degenerate
forward primer corresponding to nt 1147 to 1166 of vsp H7
and covering a relatively conserved vsp region (see
Discussion) and a reverse primer that was complementary to nt 1636 to
1653 of the vsp H7 coding sequence (Fig. 1A, step 4, and B).
The latter encodes part of the highly conserved transmembrane domain of
the surface protein. A preevaluation of the PCR on serial dilutions of
genomic DNA from G. lamblia clone GS/M-83-H7 demonstrated
that use of the degenerate forward primer generated significant amounts
of PCR amplicons only in the presence of relatively high template DNA concentrations (data not shown). Accordingly, we had to introduce a
preceding 5' RACE-PCR step (Fig. 1, step 3.1), which nonspecifically increased the amount of template cDNA and thus provided the basis for
successful PCR amplification of vsp gene sequences (Fig. 1A, steps 3.2 and 4).
In all samples investigated, the multistep PCR provided final cDNA
amplification products which migrated in agarose gels as diffuse bands
of around 550 bp (data not shown). RFLP analysis using AluI
for digestion of cDNA amplicons revealed alterations of the banding
patterns which correlated with the in vivo (Fig. 2A) and in vitro antigen switching (Fig.
2B) of the parasite. This indicated that the composition of the
vsp gene sequences within the amplification products had
changed as a consequence of the antigenic-diversification process.

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FIG. 2.
RFLP analysis of PCR-amplified vsp cDNAs.
Banding patterns (2% agarose gel) of AluI-digested cDNA
amplicons derived from RNA extracted from trophozoites recovered from
infected mice on days 0 (lane 1), 7 (lane 2), 14 (lane 3), 21 (lane 4),
and 42 (lane 5) p.i. (A) or from untreated (lane 1) or MAb
G10/4-treated (lane 2) trophozoites grown in axenic culture (B) are
shown. Size markers are shown at left.
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Amplified cDNA molecules were cloned into plasmid vector pGEMeasy, and
12 clones from each sample group were further investigated. DNA
sequence analysis indicated that all of the cloned cDNA inserts represented vsp gene segments. The open reading frames from
the cloned cDNA molecules encoded 29 peptide sequences, which
all included a combination of typical structural elements of the
C-terminal regions of the G. lamblia VSPs: (i) a high
content of cysteine residues, (ii) the presence of at least three
four-amino-acid CXXC motifs, and (iii) a hydrophobic segment
corresponding to the highly conserved transmembrane portion located at
the very C terminus of these surface proteins. The amino acid sequences encoded by cDNA clones H7, 7b, 14a, 14c, IVc, 14e, 14d/21b/IVb, IVf, 21a, IVd, and IVh (Fig. 3) also
contained a four-amino-acid GGCY motif, which may be taken as an
additional marker sequence of the VSP family (22).

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FIG. 3.
Aligned amino acid sequences representing the C-terminal
segments of VSP H7 (H7) and subvariant VSPs derived from G.
lamblia clone GS/M-83-H7. Sequences were deduced from cloned
vsp cDNA derived from parasite populations recovered on
days 0 (cDNA clones H7 and 0a), 7 (clones H7 and 7a to -c), 14 (clones
14a to -g), 21 (clones 21a to -f), and 42 (clones 42 a to -e) p.i.
from infected mice or clones IVa to -h from negatively selected axenic
trophozoites. Similarities between VSP H7 and the corresponding
segments of the subvariant VSPs identified ranged between 83 (clone 7b)
and 31% (clone 42d). Black, invariant positions; boxes, regions of
high sequence similarities (conserved residues are in boldface); dots,
alignment gaps; asterisks, positions with frequent occurrence of
cysteines (C); bar, highly conserved transmembrane segment (positions
139 to 159 of VSP H7). GenBank accession numbers are at the right.
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Molecular analysis of vsp gene expression during in
vitro antigen switching of the parasites.
A sequence comparison
that focused on those cloned vsp cDNA molecules that
originated from parasite populations before and after in vitro antigen
switching provided the following results (Fig. 3). For the original
G. lamblia clone GS/M-83-H7 culture, 11 of the 12 cDNA
clones encoded a 159-residue C-terminal sequence identical to that of
VSP H7. This result indicated predominant expression of gene vsp
H7 in clone GS/M-83-H7 prior to antigen switching. In contrast,
analogous analysis of the axenic parasite population that had been
selected against MAb G10/4-reactive trophozoites (see above) yielded a
set of eight distinct vsp cDNAs, which all encoded VSP amino
acid sequences different from that of VSP H7 (Table
1 and Fig. 3). These findings
demonstrated that elimination of MAb G10/4-reactive trophozoites left
variants whose progeny exhibited a strong diversification in
vsp gene expression.
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TABLE 1.
Prevalence of different vsp gene sequences
within sets of 12 cloned cDNA inserts derived from different tested
Giardia populations
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Molecular analysis of vsp gene expression during in
vivo antigen switching of the parasites.
Examination of the cloned
cDNA that was derived from the different parasite populations
consecutively emerging during a G. lamblia GS/M-83-H7
infection in mice provided the following results (Table 1 and Fig. 3).
G. lamblia GS/M-83-H7 trophozoites sampled at day 7 p.i. yielded transcripts that were predominantly from vsp
H7, as evidenced by detection of 9 vsp H7 versus 3 non-vsp H7 cDNA clones among 12 clones examined. Trophozoite
populations consecutively sampled later in the infection exhibited
concomitant expression of at least 7 (day 14 p.i.), 6 (day 21 p.i.), or 5 (day 42 p.i.) individual vsp genes that
were different from vsp H7. The sets of vsp genes
expressed at the different stages of infection were rather distinct
from each other and contained only one common vsp cDNA (14d
and 21b), which appeared within sample groups from days 14 and 21 p.i. The same cDNA was also found within the repertoire of cloned
vsp amplification products from axenic trophozoites that had
been negatively selected for antigenic variants (clone IVb).
Taken together, these data indicate that antigenic variation of
G. lamblia clone GS/M-83-H7 during an infection in mice
leads to the consecutive expression of relatively distinct and rather heterogeneous sets of vsp genes. Both diversification of
vsp gene expression after day 7 p.i. and the relative
individual patterns of vsp gene expression at the different
experimental time points of infection were confirmed by RFLP analysis
using AluI for digestion of corresponding cDNA amplicons
(Fig. 2A). In this analysis, the DNA banding pattern significantly
changed after day 7 p.i. and showed distinct features at the
different time points after antigen switching had occurred. Analogous
results were achieved by digestion of amplicons with another set of
frequently cutting DNA restriction enzymes (data not shown).
Determination of the genetic relationship between the different
subvariant-type VSP sequences.
Alignment of the amino acid
sequences indicated that the similarities between VSP H7 (expressed by
the parent G. lamblia GS/M-83-H7 line) and the corresponding
regions from the subvariant VSPs ranged between 83 (clone 7b) and 31%
(clone 42d) (Fig. 3). Loss of similarity of the subvariant VSP
sequences related to VSP H7 did not quantitatively correlate with the
time points at which the variants appeared during infection.
Furthermore, subvariant VSP sequences identified within the negatively
selected axenic trophozoites were heterogeneously distributed within
the entire panel of VSP sequences analyzed.
A comparison of the identified VSP sequences with corresponding regions
from other VSP sequences available in GenBank also demonstrated this
heterogeneous distribution (Fig. 4). This
comparison included all representative VSP sequences which were
available from GenBank and which had previously been used for the
definition of VSP-based phylogenetic assemblage groups A and B
(14). From the other assemblages (C and D) previously
characterized (13), no comparable VSP sequences could be
found in the database. In the comparative analysis, the majority of
GS/M-83-H7 subvariant VSP sequences were clustered in the same group
(assemblage B) as VSP H7 (Fig. 4). However, four VSP sequences
identified in the present study were as distinct from the majority of
the assemblage B-derived sequences as were vsp sequences
derived from assemblage A isolates, e.g., in vitro-derived cDNA clone
IVg and in vivo-derived clones 14f, 21e, and 42d.

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FIG. 4.
Phylogenetic relationship inferred from the C-terminal
amino acid sequences of VSP H7, the subvariant VSPs identified in this
study from cDNA, and corresponding VSP sequences available from
GenBank. Segments corresponding to residues 29 to 159 (the C-terminal
segment) of VSP H7 (Fig. 3) were aligned using ClustalX, version 1.8, and the resulting distance data were viewed using TreeView, version
1.6.1. Accession numbers of VSP sequences obtained for
GS/M-83-H7-derived G. lamblia populations are given in
Fig. 3. Those of the heterologous VSPs are as follows: AAD28789
(VSP417-4a/A-II), AAF31772 (VSP417-7/A-II), AAD04339 (VSP417-4b/A-II),
AAF04387 (VSP417-7/A-I), AAG16629 (VSP9B10/A-I), A48579 (TSP11/A-I),
AAD03497 (VSP417-3/A-II), AAD28790 (VSP417-2/A-II), AAD05040
(TSA417/A-II), A35502 (VSP9B10-LIKE), and AAF02907 (VSP417-6/A-I). Bar,
index of dissimilarity (0.1 units) among the different sequences.
Ellipses enclose sequences derived from the GS/M-83-H7 isolate
(assemblage B) or from isolates belonging to genetic assemblage A and a
third group of sequences apparently lying outside these assemblages.
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DISCUSSION |
In the past, multiple studies addressed the ability of G. lamblia to alter its VSP surface coat. Experimental infections in rodents as well as in vitro studies revealed that exposure of G. lamblia trophozoites to VSP-specific antibodies can result in a
change of the variant-type composition (reviewed in references 16 and
18). These studies were mostly based on biochemical or
immunobiochemical methods and did not evaluate in detail molecular aspects of the antigenic diversification process. In the present study,
we have for the first time extensively explored at the transcriptional
level the nature of in vitro and in vivo antigenic variation of
G. lamblia using well-characterized clone GS/M-83-H7 as the
model parasite.
The most important goal of our study was to generate data which
characterized vsp gene expression during an experimental
G. lamblia GS/M-83-H7 infection in mice. Our previous
investigations performed with the mother-offspring mouse model had
shown that antigenic variation of the parasite is initiated by
selection of variant trophozoites mediated through a direct
parasiticidal effect of lactogenic immunoglobulin A against the
parental VSP H7-type trophozoites (32). Furthermore, a
strong fluctuation of the parasite burden during the initial stages of
antigen switching had indicated that the original VSP H7 trophozoites
were eliminated and replaced by a second variant-type population. The
variant-type formation during this process of antigenic variation was
investigated in various studies (reviewed in references 16 and 18).
However, detailed biochemical and/or immunobiochemical analysis
monitoring surface antigen alterations of G. lamblia
variants emerging within clone GS/M-83-H7 mostly relied on procedures
which included a step of in vitro cultivation of the parasite (2,
7, 24). Since cultivation of subvariant types from G. lamblia clone GS/M-83-H7 had been shown to gradually change the
variant-type pattern within the parasite population (24,
30), results from experimental infections involving a subsequent
in vitro proliferation step may distort the real in vivo situation
regarding variant-type diversification.
In the present study, we chose a PCR approach (Fig. 1) which avoided
the possibility of those experimental constraints listed above. Our aim
was to isolate different vsp cDNA molecules that were
derived from G. lamblia clone GS/M-83-H7 before and after antigenic diversification. The PCR strategy relied on the use of
primers which targeted conserved regions in vsp genes and
thus might be expected to simultaneously amplify most of the sequences from the vsp gene repertoire. The pair of primers finally
used was deduced from the highly conserved 3'-terminal region of
vsp H7, which encodes the C-terminal transmembrane domain
(primer vsp-rev1; Fig. 1B), and an upstream region of
vsp H7 (target of primer vsp-forward; Fig. 1B),
which in a sequence comparison study using data from GenBank (M. Bienz,
unpublished data) had been assessed to be relatively similar to those
of vsp genes from heterologous G. lamblia
isolates. However, in applying this experimental strategy, we had to
take into account that selective processes during both the first-strand
cDNA synthesis and the different cDNA amplification steps (e.g., caused
by inefficient priming, or lack of priming, of vsp-rev1
and/or degenerate vsp-forward to a subset of vsp
genes) provided results which did not quantitatively reflect the
vsp gene expression within the different parasite
populations. Accordingly, and also because of statistical limitations
due to only having sequenced 12 cloned cDNAs per experimental group,
our investigation represents a semiquantitative approach, which could
not assess the entire complexity related to antigenic variation of
G. lamblia clone GS/M-83-H7.
By applying this PCR-based strategy, we were able to demonstrate that
(i) GS/M-83-H7 trophozoites, prior to in vitro and in vivo antigen
switching, preferentially express vsp H7 and predominantly synthesize VSP H7 (as shown by the immunofluorescence assay), (ii) in
vitro and in vivo antigen switching of the parasite is associated with
a strong diversification in vsp gene expression, (iii) in
vivo antigenic variation involves at least 22 vsp genes, and
(iv) in vivo antigenic variation represents a continuous process which
generates consecutively emerging and rather distinct variants that
exhibit, at the population level, a highly heterogeneous vsp
gene expression.
In the present study, comparative sequence analysis demonstrated high
similarities between VSP H7 and the different subvariant VSPs
identified and related most of these sequences to the same phylogenetic
assemblage (assemblage B). This close relationship of most
GS/M-83-H7-derived VSP sequences further supported a previous hypothesis suggesting that the formation of the different
Giardia vsp gene repertoires is the consequence
of ancestral combinatorial processes that included serial gene
duplication, mutation and/or recombination events within the
individual isolates (4, 35, 36). However, since four
vsp cDNA clones identified turned out to be rather distinct
from the assemblage B-type vsp gene sequences, this
ancestral gene duplication model may be insufficient to explain the
entire complexity of the evolutionary vsp gene
diversification within G. lamblia.
 |
ACKNOWLEDGMENTS |
We acknowledge A. Hemphill for carefully reading the manuscript
and T. E. Nash (NIH, Bethesda, Md.) for his gift of MAb G10/4 and
G. lamblia clone GS/M-83-H7.
This work was supported by grants obtained from the Swiss National
Science Foundation (no. 31-49439.96 and 31-58973.99).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Parasitology, P.O. Box 8466, CH-3001 Berne, Switzerland. Phone: (4131) 6312474; Fax: (4131) 6312622; E-mail:
nmueller{at}ipa.unibe.ch.
Editor:
W. A. Petri Jr.
 |
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Infection and Immunity, September 2001, p. 5278-5285, Vol. 69, No. 9
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.9.5278-5285.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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